Hybrid male sterility factors are the key to understanding speciation in animals and the molecular basis of Haldane's rule that impaired reproduction of the heterogametic sex is the first stage in reproductive isolation. The Dobzhansky-Muller model for the origin of reproductive isolation assumes that separated conspecific populations undergo independent gene substitutions which, when combined together in the hybrid, result in hybrid sterility or inviability. Virtually nothing is known about the genetic or molecular basis of hybrid incompatibility factors. A succession of sex-ratio meiotic drive systems and their suppressors that remodel spermatogenesis has been suggested as one mechanism associated with hybrid male sterility in Drosophila. In this amended proposal, we focus on the refined genetic mapping, positional cloning, sequencing, molecular analysis and evolutionary studies of at least 5-6 hybrid male sterility factors on the right arm of chromosome 3. These have each been mapped to approximately 1-2 cM regions using genetically marked P-element transgenes present at many different locations in chromosome 3 of D. mauritiana. These marked genomic regions have been introgressed into a standard genetic background of D. simulans and the homozygous introgression hybrids tested for fertility. Across the third chromosome, 19 factors are associated with hybrid male sterility in suitable genetic backgrounds. One of these factors, denoted tmy, is associated not only with severely reduced hybrid male fertility but also with sex ratio meiotic drive. Using a novel genetic mapping strategy that allows easy phenotypic identification of recombinants with an exchange in the region of interest, the tmy gene has been mapped to a region of 10 kb or smaller. This same mapping strategy will be used to refine the map positions of the other hybrid male sterility factors, which will have sufficient resolution to support positional cloning. Each hybrid male sterility factor will be cloned and sequenced and its molecular organization determined, and if feasible will be used for germline transformation rescue. The tmy system will be analyzed in detail cytologically. Preliminary evidence suggests a high rate of second-division Y chromosome loss and fragmentation. Downstream functional analysis of the other hybrid male sterility factors is beyond the scope of the present proposal but is of course a long-range goal. Each of the 5-6 hybrid male sterility factors that will be isolated will be subjected to an evolutionary analysis for its rate and pattern of molecular evolution, using phylogenetic analysis, polymorphism and divergence analysis between D. melanogaster, D. simulans and D. mauritiana, and intrapopulation analysis of polymorphisms and haplotypes to look for evidence of recent selective sweeps.